Packaging systems for human recombinant adenovirus to be used in gene therapy

Abstract

Presented are ways to address the problem of replication-competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector are suited for large scale production of recombinant adenoviruses. A described system produces replication-defective adenovirus. The system includes a primary cell containing a nucleotide derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleotide is derived from an adenovirus, has at least one functional encapsidation signal and at least one functional Inverted Terminal Repeat, and lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication-competent adenovirus in the primary cell into which the isolated recombinant nucleotide is to be transferred.

1 . An isolated first nucleic acid molecule comprising genetic information encoding functional adenovirus E1A, E1B 21 kDa and E1B 55 kDa gene products, wherein said first nucleic acid molecule lacks genetic information from an adenovirus pIX gene that can mediate homologous recombination with a second nucleic acid molecule having genetic information encoding a functional or active adenovirus pIX gene product, and further wherein said first nucleic acid molecule lacks genetic information encoding adenovirus proteins other than proteins encoded by adenovirus early region 1. 
     
     
         2 . The isolated first nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule comprises nucleotides 459-3510 of Ad5. 
     
     
         3 . The isolated first nucleic acid molecule of  claim 2 , wherein the isolated nucleic acid molecule lacks nucleotides from the E1 region of Ad5 downstream of nucleotide 3510. 
     
     
         4 . The isolated first nucleic acid molecule of  claim 1 , further comprising a promoter operatively linked to at least the genetic information encoding the functional E1A gene product. 
     
     
         5 . The isolated first nucleic acid molecule of  claim 4 , wherein the promoter comprises a constitutive promoter. 
     
     
         6 . The isolated first nucleic acid molecule of  claim 5 , wherein the constitutive promoter comprises a PGK promoter. 
     
     
         7 . The isolated first nucleic acid molecule of  claim 4 , wherein the promoter comprises a human PGK promoter. 
     
     
         8 . The isolated first nucleic acid molecule of  claim 1 , further comprising the Hepatitis B Virus polyadenylation signal located downstream of an E1B stop codon. 
     
     
         9 . The isolated first nucleic acid molecule of  claim 4 , further comprising the Hepatitis B Virus polyadenylation signal located downstream of an E1B stop codon. 
     
     
         10 .- 18 . (canceled) 
     
     
         19 . An isolated adenovirus packaging cell comprising the isolated first nucleic acid molecule of  claim 1 . 
     
     
         20 . The isolated adenovirus packaging cell of  claim 19 , wherein the cell is a mammalian cell. 
     
     
         21 . The isolated adenovirus packaging cell of  claim 19 , wherein the cell is a human cell. 
     
     
         22 . The isolated adenovirus packaging cell of  claim 19 , wherein the cell is a diploid cell. 
     
     
         23 . The isolated adenovirus packaging cell of  claim 19 , wherein the cell is derived from an embryonic cell. 
     
     
         24 . The isolated adenovirus packaging cell of  claim 23 , wherein the embryonic cell is selected from the group consisting of kidney cells, lung cells, and retinoblast cells. 
     
     
         25 . The isolated adenovirus packaging cell of  claim 24 , wherein the embryonic cell is a retinoblast cell. 
     
     
         26 . The isolated adenovirus packaging cell of  claim 19 , wherein the cell is immortalized. 
     
     
         27 . A method for making an isolated adenovirus packaging cell comprising introducing into the cell the isolated first nucleic acid molecule of  claim 1 . 
     
     
         28 . The method according to  claim 27 , further comprising integrating the isolated first nucleic acid molecule into the precursor cell's genome. 
     
     
         29 .- 37 . (canceled)